Quantification of miR-182 Expression

Total RNA was isolated using a miRNeasy mini kit (Qiagen) and reverse-transcribed into cDNA with the SuperScript III kit (Life Technologies) as described previously [26 (link)]. For miR-182 expression analysis, cDNA was synthesized from total RNA with the TaqMan Reverse Transcription kit (Applied Biosystems) with specific primers and the cDNA was subjected to TaqMan Probe-based Real-Time PCR using TaqMan miRNA assays Universal PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. Real-time reverse transcription-polymerase chain reaction (RT–PCR) was performed with SYBR Green (Applied Biosystems) using a QuantStudio 7 PCR System. The expression levels of miRNA were calculated as the amount of target miRNA relative to that of RNU48 control to normalize the initial input of total RNA.